1. Field of the Invention
This invention relates to methods for modifying nucleic acid molecules for their later manipulation and, more particularly, this invention relates to methods for labeling modified nucleic acid molecules or for immobilizing modified nucleic acid molecules on to specified supports.
2. Background of the Invention
Manipulating nucleic acids for subsequent use and analysis is increasing in importance. For example, the labeling of polynucleotides for later use as indicators of complementary sequences in target molecules has wide utility in diagnostic and research applications. Also, designing polynucleotides to interact with inert substrates such as activated polyacrylamide gels will increase the utility of labeling and sequencing activities.
Present techniques for labeling and/or immobilizing nucleic acids have several drawbacks. Labeling processes will be discussed first.
Three widely-accepted methods exist for the labeling of molecules: radio-labeling, fluorescent-labeling, and chemiluminescent labeling. There are benefits to each method. Radio-labels are more sensitive. Fluorescent labels are safer, can be detected in real time, and several different labels can be simultaneously monitored. Chemiluminescent labels have the same sensitivity as radio-labels, and are also safer, but can only be used once.
The most commonly used process for fluorescent labeling of nucleic acids involves enzymatic reactions. Typically, organic fluorophores are first chemically introduced into PCR oligonucleotide primers or nucleoside triphosphates and then incorporated by using DNA or RNA polymerases (via the use of polymerase chain reaction processes, or terminal polynucleotide transferase). These techniques are expensive and not always efficient and reliable. Also, some labelling processes result in obfuscating or breaking the continuity of a target oligonucleotide sequence on the labeled molecule during polymerase reactions. As such, less accurate duplexes form when sequencing DNA strands via hybridization procedures.
As noted above, methods for immobilizing nucleic acids and related molecules also have drawbacks. Oligonucleotides can be immobilized by reacting their 3'-dialdehyde termini (produced through chemical treatment with the oligos) with hydrazide groups located on gel support compounds. Unfortunately, typical hydrazide chemistry protocols require preactivation of oligonucleotides, and also result in attachment bonds which are not resistant to repeated hybridization processes.
A need exists in the art to provide modified nucleotides which can interact with labels and high volume supports. A need also exists to provide efficient and inexpensive methods to label and otherwise examine synthetically-derived, or naturally-occurring nucleotide molecules of any length. The methods and resulting modified molecules must be compatible with hybridization occurrences between the labeled polynucleotide and non-labeled polynucleotides. The methods also should allow for the incorporation of different labels into a nucleic acid fragment for multi-color hybridization analysis. Lastly, the methods should facilitate chemical coupling, or photopolymerization of modified molecules, such as polynucleotides, with high volume supports such as gel substrates.